Project Summary/Abstract We have recently discovered that rat, mouse, and human dopamine transporters (DATs) are modified by palmitoylation, the covalent attachment of palmitic acid. This is the first demonstration of lipid modification for DAT or related neurotransmitter transporters, and significantly impacts our view of transport regulatory mechanisms. Palmitoylation is reversible and dynamic, conferring the ability of proteins to respond to physiologic signals and participate in regulatory processes in a manner analogous to phosphorylation. Our preliminary studies indicate that palmitoylation strongly regulates dopamine (DA) transport capacity and opposes DAT degradation. These novel findings suggest that palmitoylation has high potential to impact dopaminergic signaling in both short- and long-term manners that may be relevant to dopaminergic dysregulation in drug abuse and neurologic disease states. Preliminary studies indicate that methamphetamine can modulate DAT palmitoylation in rats, which may impact acute and chronic regulation of the transporter. In addition, we have identified two palmitoylation sites in rat DAT, with early results consistent with different functions attributable to modification of the different sites. Many major questions related to DAT palmitoylation remain to be addressed. At the most basic level these include characterizing psychostimulant drug effects on DAT palmitoylation in vivo and determining functions associated with each of the palmitoylation sites identified, for both rat and human transporters. Our long-term goal is to understand the regulatory processes involved in modulating DAT activity and to establish causative links in dysfunction of these processes to human neurological disorders and drug abuse. The objective of this proposal is to perform fundamental analyses of the role of palmitoylation in modulation of rat DAT function that will lay the groundwork for future studies relevant to understanding DAT activity in normal and pathophysiologic conditions. Our central working hypothesis is that palmitoylation of DAT increases DA transport kinetics and decreases DAT degradation, thereby functioning as a mechanism that would lead to increased DAT transport capacity. To test this hypothesis we propose two specific aims: 1) Assessment of transporter function associated with individual palmitoylation sites; and 2) Assessment of acute psychostimulant drug treatments on the DAT palmitoylation state. For the first aim we will employ site-directed mutagenesis to generate single or double palmitoylation site mutants and analyze each for DAT function and regulation. For the second aim we will treat rats with psychostimulant drugs and measure the time-course and duration of changes in DAT palmitoylation and DA uptake capacity. This significant research will be performed by undergraduate and graduate students and is expected to substantially advance our understanding of the role of this previously unknown lipid modification on DAT function, and may provide insights into dysregulation of DAT activities leading to dopaminergic disease or drug abuse.